CN114504660A - Magneto-optical dual-field contrast agent and preparation method and application thereof - Google Patents

Abstract

The invention relates to the technical field of material science and biomedical science and technology, in particular to a magneto-optical double-field contrast agent, a preparation method and application thereof, wherein the structure of the contrast agent is a superparamagnetic ferroferric oxide structure coated by hyaluronic acid-coated mesoporous silica; meanwhile, rare earth elements are filled in the superparamagnetic iron tetroxide structure coated by the mesoporous silica. On the basis of controllable and accurate positioning magnetic field radiography, the magneto-optical dual-field contrast agent is doped with trace amounts of low-toxicity rare earth luminescent material baits to prepare the contrast agent, so that the biotoxicity of the contrast agent is further reduced, the optical coherence imaging and the magnetic field radiography dual-field imaging are realized, the developing effect is enhanced, the stenosis degree on the coronary artery lumen anatomical structure can be accurately judged, and the pathological and physiological change degree of the contrast agent is predicted.

Description

A kind of magneto-optical dual-field contrast agent, preparation method and application thereof

technical field

The invention relates to the technical fields of material science and biomedical science and technology, in particular to a magneto-optical dual-field contrast agent and a preparation method and application thereof.

Background technique

The information disclosed in this Background section is only for enhancement of understanding of the general background of the invention and should not necessarily be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person of ordinary skill in the art.

At present, the latest generation of intravascular angiography on the market uses optical coherence imaging technology. However, due to factors such as the complex intravascular fluid environment, the imaging effect and efficiency are not high, which seriously affects the application scope of this technology in intravascular angiography. For critical coronary lesions, coronary angiography only indicates the degree of stenosis on the anatomical structure of the coronary lumen, and the degree of pathological and physiological changes cannot be predicted. Coronary angiography alone cannot accurately quantify lesions, and cannot accurately assess stent expansion and apposition. Moreover, due to the high incidence of stent underexpansion, poor apposition, and dissection, it may lead to an increased risk of stent thrombosis and stent restenosis. Light field angiography can obtain a clear vascular wall structure and lumen plan, which is of great significance for the identification and measurement of coronary lesions, guidance of surgical strategies, and optimization of stent effects.

In upper gastrointestinal angiography, traditional barium meal angiography cannot show the specific morphological features of the upper gastrointestinal tract; lower gastrointestinal lesions mainly rely on white light endoscopy combined with pathological biopsy of abnormal intestinal mucosa to diagnose polyps, tumors, inflammation or vascular malformations etc., but the inspection effect of white light endoscopy is far from ideal, and it is reported that standard electronic colonoscopy even has a missed diagnosis rate of up to 40% of adenomas. Therefore, how to solve the above problems becomes the key.

SUMMARY OF THE INVENTION

In order to solve the above problems existing in the prior art, the present invention provides a magneto-optical dual-field contrast agent and a preparation method and application thereof. Using the magneto-optical dual-field contrast agent, on the basis of controllable and precise positioning of magnetic field contrast, doping A small amount of low-toxic rare-earth luminescent material bait is made into a contrast agent to further reduce its biological toxicity, and at the same time, it realizes dual-field imaging of optical coherence imaging and magnetic field angiography, enhances the imaging effect, and can accurately determine the degree of stenosis on the anatomical structure of the coronary artery lumen. , and predict the degree of pathological and physiological changes.

Specifically, the technical solution of the present invention is as follows:

In the first aspect of the present invention, a magneto-optical dual-field contrast agent, the structure of the contrast agent is a superparamagnetic iron tetroxide structure coated with hyaluronic acid and mesoporous silica; The silicon oxide-coated superparamagnetic iron tetroxide structure is also filled with rare earth elements.

In a second aspect of the present invention, a method for preparing a magneto-optical dual-field contrast agent, comprising:

(1), prepare superparamagnetic iron tetroxide: add ammonia water quickly to the mixed solution of FeCl ₃ and FeCl ₂ , continue the reaction and finish and be cooled to room temperature, and obtain superparamagnetic iron tetroxide after cleaning;

(2), the preparation of mesoporous silica coating layer: stirring and mixing cetyl trimethyl ammonium bromide, absolute ethanol, deionized water, adding ammonia solution and superparamagnetic ferric tetroxide simultaneously, After stirring, wait for a period of time and continue to add ethyl orthosilicate dropwise, and the mixture is heated in a water bath for the first time to obtain a precipitate; the precipitate is centrifuged to obtain a secondary precipitate, the secondary precipitate is washed, and the supernatant is poured out. Then NH ₄ NO ₃ and ethanol solution were added and stirred, and the mixed solution was heated in a water bath for the second time to obtain three precipitates. After standing for a period of time, the supernatant liquid was poured out and washed; the final turbid liquid was Drying is carried out to finally obtain a superparamagnetic iron tetroxide structure coated with mesoporous silica;

(3) Mesoporous filling with rare earth elements: put the prepared mesoporous silica-coated superparamagnetic ferric oxide structure and erbium nitrate solution in a vortex mixer and fully mix them and dry them. Finally, a superparamagnetic ferric oxide doped rare earth element structure coated with mesoporous silica is obtained;

(4), mixing the product prepared in (3) with hyaluronic acid and water to obtain a magneto-optical dual-field contrast agent.

In a third aspect of the present invention, the magneto-optical dual-field contrast agent and/or the contrast agent obtained by any of the preparation methods of the magneto-optical dual-field contrast agent is used in magnetic resonance imaging.

In the fourth aspect of the present invention, the application of the magneto-optical dual-field contrast agent and/or the contrast agent obtained by any of the preparation methods of the magneto-optical dual-field contrast agent in coronary intravascular high-definition angiography.

In a fifth aspect of the present invention, the magneto-optical dual-field contrast agent and/or the contrast agent obtained by any of the preparation methods of the magneto-optical dual-field contrast agent is used in hemodynamic evaluation.

One or more technical solutions in the present invention have the following beneficial effects:

(1) Using rare earth luminescent materials as intravascular contrast agents to achieve the enhancement of optical imaging in blood vessels, especially in capillaries. The luminescent materials prepared by rare earth ion bait have excellent luminescent properties; the incorporation of hyaluronic acid enhances biocompatibility Intravascular real-time hemodynamic assessment. Help clinical realization of new functional assessment and microcirculation. Optical coherence imaging combined with superparamagnetic iron tetroxide magnetic field angiography can achieve precise positioning, which can safely and effectively evaluate and treat coronary artery lesions. Optical coherence imaging not only confirms the absence of severe anatomical stenosis in most patients, but also identifies unstable coronary lesions. Magneto-optic dual-field diagnosis of coronary heart disease can improve the diagnostic sensitivity, negative predictive value, accuracy, and enhance the diagnostic efficiency.

(2) High-efficiency gastrointestinal angiography is achieved by oral administration of mesoporous silica-loaded contrast agents with ferric oxide nanoparticles. Its mechanism of action is to reduce T1 and T2 relaxation times, and it is mainly used in various medical applications of magnetic resonance imaging. Application to display the specific morphological characteristics of the digestive tract. The use of optical coherence imaging can clearly image the mucosa and submucosa, and can observe the blood vessels in the submucosa, identify benign polyps and adenomas, reduce the cost of pathological examination, and discard the risk of malignant lesions during colonoscopy. to a minimum.

(3) Cells phagocytose the magneto-optical dual-field contrast agent of superparamagnetic ferric oxide doped with rare earth luminescent material bait wrapped with mesoporous silica and then use the magnetic field to deliver it to the tumor site more accurately, so that macrophages carry the contrast agent The agent is enriched around the tumor. Under magnetic resonance imaging, the outer edge of the tumor and the interior of the tumor form a contrast of light and dark, which facilitates the imaging observation of the tumor. At the same time, the near-infrared light emitted by the rare earth luminescent material is used to control the release of drugs/other active substances to achieve the purpose of treating tumors, which is developing from tumor diagnosis to tumor treatment. Due to the outstanding physical properties of mesoporous silica and mixed with hyaluronic acid, the biocompatibility is improved, and the contrast agent is made with low-toxic rare earth luminescent material bait, which can not only meet the requirements of optical coherence imaging, but also make high-concentration colloids Stable and rapid renal clearance.

Description of drawings

The accompanying drawings forming a part of the present invention are used to provide further understanding of the present invention, and the exemplary embodiments of the present invention and their descriptions are used to explain the present invention, and do not constitute an improper limitation of the present invention.

FIG. 1 is a schematic structural diagram of the superparamagnetic triiron tetroxide doped rare earth luminescent material wrapped with mesoporous silica in Example 1. FIG.

FIG. 2 is a schematic diagram of the synthesis steps of the mesoporous silica-wrapped superparamagnetic iron tetroxide-doped rare-earth light-emitting material of Example 1. FIG.

FIG. 3 is the verification of the cytotoxicity of the contrast agent in Example 3. FIG.

FIG. 4 shows the results of the TY stomach experiment described in Example 3. FIG.

Scanning after taking TY 500ml, the filling is good, and the stomach wall and mucous membrane are clearly displayed.

Fig. 5 is the TY ultrasonic in vitro water bladder experiment described in embodiment 3;

The TY 100ml ultrasound scan was added to the abnormal water bladder, and the echo was uniform without obvious artifacts.

Fig. 6 is the TY ultrasound stomach experiment described in embodiment 3;

After taking TY 500ml, the ultrasound scan after 10 minutes clearly shows the structure of gastric antrum, duodenum and retroperitoneum.

Fig. 7 is the TY magnetic resonance stomach experiment described in embodiment 3;

MRI scan after taking TY 1000ml clearly shows gastric structure and mucosa.

FIG. 8 is a SEM photograph of the contrast agent sample described in Example 3. FIG.

FIG. 9 is a room temperature hysteresis loop of the contrast agent sample described in Example 3. FIG.

Detailed ways

It should be noted that the following detailed description is exemplary and intended to provide further explanation of the invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

It should be noted that the terminology used herein is for the purpose of describing specific embodiments only, and is not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly dictates otherwise, the singular is intended to include the plural as well, furthermore, it is to be understood that when the terms "comprising" and/or "including" are used in this specification, it indicates that There are features, steps, operations, devices, components and/or combinations thereof.

At present, the existing contrast agents cannot accurately quantify lesions, and cannot accurately assess stent expansion and adherence. Moreover, due to the high incidence of stent underexpansion, poor adherence, and dissection, it may lead to the risk of stent thrombosis and stent restenosis. Increase. Moreover, there is a high rate of missed diagnosis. To this end, the present invention provides a magneto-optical dual-field contrast agent and a preparation method and application thereof.

In one embodiment of the present invention, a magneto-optical dual-field contrast agent, the structure of the contrast agent is a superparamagnetic iron tetroxide structure coated with hyaluronic acid and mesoporous silica; The porous silica-coated superparamagnetic iron tetroxide structure is also filled with rare earth elements.

In view of the problems that the contrast agents on the market have poor imaging effects in coronary arteries and digestive tracts, cannot fully meet clinical needs, and cause damage to target organs and kidneys, the use of superparamagnetic iron tetroxide to achieve controllable and precise positioning magnetic field angiography On the basis of , the contrast agent doped with a trace amount of low-toxic rare earth luminescent material bait further reduces its biological toxicity, and at the same time realizes the dual-field imaging of optical coherence imaging and magnetic field angiography, which enhances the development effect and can accurately determine the coronary lumen. Anatomical stenosis degree, and predict the degree of pathological and physiological changes.

Mesoporous silica has outstanding physical properties, increases the stability of developing functional materials, and is mixed with hyaluronic acid to improve biocompatibility. The magneto-optical dual-field angiography doped with rare-earth luminescent elements overcomes the technical defects of long imaging time and blood flow blocking, and at the same time solves the poor penetration of traditional optical angiography, which cannot well observe large or deep plaques. Problems, can clearly distinguish the mucosa, submucosa and muscularis propria, so as to determine the lesion.

In one embodiment of the present invention, the rare earth material is erbium.

At present, gadolinium is widely used in contrast agents, but due to its strong toxicity, it is easy to cause adverse reactions and postoperative target organ damage, and the contrast agent can cause damage to the kidneys and microcirculation. The new contrast agent uses low-toxicity rare earth luminescent elements ——Bait, superparamagnetic ferric tetroxide is added to realize optical field and magnetic field dual-field imaging, silica coating effectively realizes hydrophilic modification, and hyaluronic acid is mixed at the same time to improve biocompatibility and greatly reduce imaging damage to the human body.

In one embodiment of the present invention, a method for preparing a magneto-optical dual-field contrast agent, comprising:

(1), prepare superparamagnetic iron tetroxide: add ammonia water quickly to the mixed solution of FeCl ₃ and FeCl ₂ , continue the reaction and finish and be cooled to room temperature, and obtain superparamagnetic iron tetroxide after cleaning;

(2), the preparation of mesoporous silica coating layer: stirring and mixing cetyl trimethyl ammonium bromide, absolute ethanol, deionized water, adding ammonia solution and superparamagnetic ferric tetroxide simultaneously, After stirring, wait for a period of time and continue to add ethyl orthosilicate dropwise, and the mixture is heated in a water bath for the first time to obtain a precipitate; the precipitate is centrifuged to obtain a secondary precipitate, the secondary precipitate is washed, and the supernatant is poured out. Then NH ₄ NO ₃ and ethanol solution were added and stirred, and the mixed solution was heated in a water bath for the second time to obtain three precipitates. After standing for a period of time, the supernatant liquid was poured out and washed; the final turbid liquid was Drying is carried out to finally obtain a superparamagnetic iron tetroxide structure coated with mesoporous silica;

(3) Mesoporous filling with rare earth elements: put the prepared mesoporous silica-coated superparamagnetic ferric oxide structure and erbium nitrate solution in a vortex mixer and fully mix them and dry them. Finally, a superparamagnetic ferric oxide doped rare earth element structure coated with mesoporous silica is obtained;

(4), mixing the product prepared in (3) with hyaluronic acid and water to obtain a magneto-optical dual-field contrast agent.

Through the above preparation method, a magneto-optical dual-field contrast agent with a unique structure and a superparamagnetic triiron tetroxide doped rare-earth luminescent material wrapped with mesoporous silica can be obtained.

In step (1), the volumes of FeCl ₃ , FeCl ₂ and ammonia water are respectively 10-30mL, 1-30mL, 4-10mL; preferably, 20mL, 20mL, 7.5mL; or, the FeCl ₃ concentration is 0.1-0.3 mol/L; preferably, 0.2 mol/L; or, the FeCl ₂ concentration is 0.05-0.2 mol/L; preferably 0.1 mol/L; or, the weight percentage of the ammonia water is 28wt%.

Among them, controlling the dosage of FeCl ₃ , FeCl ₂ and ammonia water is helpful to control the uniformity of superparamagnetic ferric oxide, and avoid causing unevenness of ferric oxide nanoparticles and reducing the analysis accuracy of the material.

In step (1), after FeCl ₃ and FeCl ₂ are mixed, stir for 5-15min, preferably 10min; or, the continuous reaction time is 15-35min, preferably, 25min; or, what is used for cleaning is sodium chloride and double distilled water, the volume ratio of sodium chloride solution and double distilled water is 1:2～3; or, the concentration of sodium chloride solution is 0.01-0.03mol/L, preferably 0.02mol/L; or, drying : The heating and drying temperature is 60℃, and the time is 1～2h.

The reaction time also has a certain influence on the morphology of the ferric oxide, and the reaction time is too long to easily lead to particle agglomeration, which is not conducive to the coating of silica and the filling of rare earth luminescent elements. Among them, controlling the volume ratio of sodium chloride solution and water is also conducive to the efficient removal of impurities.

In step (2), the mass of cetyltrimethylammonium bromide is 60-85mg, and the volume ratios of absolute ethanol and deionized water are respectively 10-20mL and 20-35mL; The mass of trimethylammonium bromide is 75mg, and the volume ratios of absolute ethanol and deionized water are 15mL and 25mL, respectively; , 0.15-0.40 g, 100-150 μl; preferably, 250 μL: 0.23 g: 129 μL.

Among them, the amount of each component plays an important role in obtaining a silicon dioxide layer with a uniform thickness.

In step (2), the concentration of the aqueous ammonia solution is 28wt%; or, the waiting time for the end of stirring is 5-15s, preferably 10s; or, the first water bath heating temperature is 40-70°C, preferably 60°C; Or, the first water bath heating time is 4-7h, preferably 6h; or, the volume of the NH ₄ NO ₃ ethanol solution is 10-30mL, preferably 20mL; or, the second water bath heating and stirring time is 1- 3h, the temperature is 50-70°C; or, cleaning: the volume ratio of absolute ethanol and deionized water is 1:2 to 3; drying: the time is 2h, and the temperature is 80°C.

The temperature and time of the two water bath treatments play an important role in obtaining higher uniformity of mesoporous silica. If the mesoporous structure is not uniform, it is easy to cause uneven filling of rare earth light-emitting elements. If the structure of the prepared material is not uniform, it is unfavorable for optical coherence imaging.

In an embodiment of the present invention, the application of the magneto-optical dual-field contrast agent and/or the contrast agent obtained by the preparation method of the magneto-optical dual-field contrast agent in magnetic resonance imaging.

In an embodiment of the present invention, the application of the magneto-optical dual-field contrast agent and/or the contrast agent obtained by the preparation method of the magneto-optical dual-field contrast agent in coronary intravascular high-definition angiography. Optical coherence imaging combined with superparamagnetic iron tetroxide magnetic field angiography can achieve precise positioning, which can safely and effectively evaluate and treat coronary artery lesions. Optical coherence imaging not only confirms the absence of severe anatomical stenosis in most patients, but also identifies unstable coronary lesions. Magneto-optic dual-field diagnosis of coronary heart disease can improve the diagnostic sensitivity, negative predictive value, accuracy, and enhance the diagnostic efficiency.

In an embodiment of the present invention, the application of the magneto-optical dual-field contrast agent and/or the contrast agent obtained by the preparation method of the magneto-optical dual-field contrast agent in hemodynamic evaluation. Rare earth luminescent materials are used as intravascular contrast agents to achieve the enhancement of optical imaging in blood vessels, especially in capillaries. The luminescent materials prepared by rare earth ion bait have excellent luminescent properties; the incorporation of hyaluronic acid enhances biocompatibility and is effective for intravascular Perform real-time hemodynamic assessments.

In order to enable those skilled in the art to understand the technical solutions of the present invention more clearly, the technical solutions of the present invention will be described in detail below with reference to specific embodiments.

Example 1

(1) Preparation of superparamagnetic iron tetroxide nanoparticles:

First, 20 mL of Fe ₂ (SO ₄ ) ₃ solution with a concentration of 0.2 mol/L was mixed with a Fe ₂ (SO ₄ ) ₂ solution with a concentration of 0.1 mol/L, and the mixed solution was mechanically stirred for 10 min under the protection of N ₂ . Then 7.5 mL (28% wt) ammonia water was injected into the mixture at one time and rapidly, and the reaction was continued for 25 min and then cooled to room temperature. Then wash with 0.02mol/L sodium chloride solution and double distilled water respectively. A test magnet was placed in the mixture, the supernatant liquid was poured out after the particles settled, and dried at 60° C. to finally obtain 0.39 g of superparamagnetic iron tetroxide nanoparticles.

(2) Preparation of mesoporous silica coating:

Add 75mg cetyl trimethyl ammonium bromide (CTAB), 15mL absolute ethanol, 25mL deionized water to the beaker in turn, stir and mix well, at the same time add 250ul (28%wt) ammonia solution and 0.23g to prepare After stirring, 129 μL (analytical grade, AR) ethyl orthosilicate (TEOS) was added dropwise for 10s after stirring, and the mixture was heated in a 60°C water bath for 6h. After the reaction, the precipitate was centrifuged at a speed of 2000 rpm. The precipitate was washed three times with absolute ethanol and deionized water, and then NH ₄ NO ₃ ethanol solution was added, stirred for 2 h, and heated in a 60°C water bath. After the end, let stand for 30min, carefully aspirate the supernatant. Repeated washing, sucking the supernatant three times, and then washed three times with absolute ethanol and deionized water, and dried the obtained turbid liquid at 80 °C for 2 h, and finally obtained 0.29 g of mesoporous silica-coated superparamagnetic trioxide tetraoxide. Powder samples of iron nanocomposite structures.

(3) Step 3: Filling Mesopores with Rare Earth Elements

The sample prepared in the previous step and 1 mL of 15 μmol/L low-toxic erbium nitrate solution were put into a vortex mixer and fully mixed for 20 min, and the turbid liquid was dried at 60° for 30 min to finally obtain 0.30 g of mesoporous silica-coated supernatant. Paramagnetic ferric oxide doped rare earth element nanocomposite structure.

(4) Step 4: Mix hyaluronic acid and deionized water

The sample prepared in the previous step was mixed with 3Kg of hyaluronic acid (99% concentration) and 297Kg of deionized water for 2 hours, and finally 300.0003Kg of mesoporous silica-wrapped superparamagnetic iron tetroxide doped rare earth luminescence was obtained. material contrast agent.

Example 2

(1) Preparation of superparamagnetic iron tetroxide nanoparticles:

First, 20 mL of Fe(NO ₃ ) ₃ solution with a concentration of 0.2 mol/L was mixed with a Fe(NO ₃ ) ₂ solution with a concentration of 0.1 mol/L, and the mixed solution was mechanically stirred for 10 min under the protection of N ₂ . Then 7.5 mL (28% wt) ammonia water was injected into the mixture at one time and rapidly, and the reaction was continued for 25 min and then cooled to room temperature. Then wash with 0.02mol/L sodium chloride solution and double distilled water respectively. A test magnet was placed in the mixture, the supernatant liquid was poured out after the particles settled, and dried at 60° C. to finally obtain 0.39 g of superparamagnetic iron tetroxide nanoparticles.

(2) Preparation of mesoporous silica coating:

Add 75mg cetyl trimethyl ammonium bromide (CTAB), 15mL absolute ethanol, 25mL deionized water to the beaker in turn, stir and mix well, at the same time add 250ul (28%wt) ammonia solution and 0.23g to prepare After stirring, 129 μL (analytical grade, AR) ethyl orthosilicate (TEOS) was added dropwise for 10s after stirring, and the mixture was heated in a 60°C water bath for 6h. After the reaction, the precipitate was centrifuged at a speed of 2000 rpm. The precipitate was washed three times with absolute ethanol and deionized water, and then NH ₄ NO ₃ ethanol solution was added, stirred for 2 h, and heated in a 60°C water bath. After the end, let stand for 30 min, and carefully aspirate the supernatant. Repeated washing, sucking the supernatant three times, and then washed three times with absolute ethanol and deionized water, and dried the obtained turbid liquid at 80 °C for 2 h, and finally obtained 0.29 g of mesoporous silica-coated superparamagnetic trioxide tetraoxide. Powder samples of iron nanocomposite structures.

(3) Step 3: Filling Mesopores with Rare Earth Elements

The sample prepared in the previous step and 1 mL of 15 μmol/L low-toxic erbium nitrate solution were put into a vortex mixer and fully mixed for 20 min, and the turbid liquid was dried at 60° for 30 min to finally obtain 0.30 g of mesoporous silica-coated supernatant. Paramagnetic ferric oxide doped rare earth element nanocomposite structure.

(4) Step 4: Mix hyaluronic acid and deionized water

The sample prepared in the previous step was mixed with 3Kg of hyaluronic acid (99% concentration) and 297Kg of deionized water for 2 hours, and finally 300.0003Kg of mesoporous silica-wrapped superparamagnetic iron tetroxide doped rare earth luminescence was obtained. material contrast agent.

Example 3

In this example, the contrast agent prepared in Example 1 is used as an example to illustrate the safety and application. It should be noted that the contrast agent prepared within the scope of the technical concept of the present invention can achieve similar effects to the following applications, with Similar to the application below.

1. Contrast agent cytotoxicity verification provided in Example 1

Experimental methods: MTT method was used to investigate the cytotoxicity of contrast agents. For experimental cells, EA.hy926 cells in logarithmic growth phase were taken, 96-well plates were taken, 5×10 ³ cells were added to each well, and they were cultured in a cell incubator containing 5% CO ₂ at 37°C. After culturing for 24 hours, the culture medium in the well was aspirated, and 100 μL of medium containing different mass concentrations (10, 20, 50, 100, 200, 500 and 1 000 μg/mL) and no contrast agent (0 concentration control group) was added to the well. , 6 replicate wells were set for each concentration, and the culture was continued for 24 hours. The culture medium was aspirated again, washed three times with PBS, and then 100 μL of MTT (1 mg/mL) was added to each well for 4 h and then removed. Add 150 μL of dimethyl sulfoxide to each well, and after shaking for 1 minute, use a microplate reader to measure and record the absorbance value at 570 nm immediately.

Experimental results: The MTT method was used to study the toxic effect of contrast agents on EA.hy926 cells. After 24 hours of incubation in culture medium containing different concentrations of contrast agent, the survival rate of EA.hy926 cells decreased with the increase of contrast agent concentration. % or more, but there was no significant statistical difference with the control group. When the concentration was increased to above 500 μg/mL, the cell viability decreased significantly, which was statistically different from that of the control group, but the cell viability was still higher than 80%, indicating that the contrast agent had good biocompatibility and could be used in the follow-up in vivo studies.

2. Contrast agent abdominal magnetic resonance MRI assessment.

Twenty male Yorkshire pigs were fasted for more than 4 hours before the test, and the lower abdomen scan was required to fast for more than 8 hours. For upper abdominal scan subjects, 30 minutes before the scan, take 500mL of contrast agent. The lower abdomen and the whole abdomen were scanned and 1000mL of contrast medium was orally administered in 4 times within 2 hours.

After taking the contrast agent, it shows that this product can stay in and fill the gastrointestinal tract for a long time, and produce a uniformly distributed hypoechoic interface area on the ultrasound image; it appears as a watery low-density shadow on CT; it appears on MRI. Long T1, T2 signals. It can effectively display the layers and structure of the gastrointestinal wall, enhance the display of retroperitoneal organs and the outline of the surrounding structures, and understand the peristalsis and emptying functions of the gastrointestinal tract. At the same time, it removes the interference of gas and mucus in the gastrointestinal cavity, effectively reduces and eliminates the artifacts and interference in the inspection area, and facilitates the determination of the location, shape, size, scope and internal structure of the lesion. The effective retention half-attenuation time in the stomach reaches 65 minutes, which ensures sufficient inspection window time. The display rate of each segment of the gastrointestinal tract is increased from an average of about 30% to 89%, and the gastric display rate is increased from 25% to 100%, which is beneficial to the liver. The outline of the left lobe edge, the duodenal filling increased from 30% to 95%, so that the pancreatic head and uncinate process can be clearly set off and displayed, the small intestine and colon filling increased from 10% to 85% on average, see figure 1. The borders of kidneys, spleen, abdominal lymph nodes and bladder were significantly improved, abdominal mass was easy to find, and ascites and mesentery were well displayed and delineated. The range of tumor infiltration and edema can be clearly displayed on the image of gastric cancer. Chi-square (χ2) test was performed using Stata medical statistical software, and P<0.0001 indicated that the two methods were significantly different, as shown in Figure 2.

Table 1 Study on the stability of TY in 90 patients with CT, MRI and magnetic resonance gastrointestinal tract

Table 2 Comparison of CT, MRI and magnetic resonance gastrointestinal display results of 90 subjects before and after using TY

CONCLUSION: The aid is safe and effective, and has a good general-purpose type. It is widely used in the contrast display of the gastrointestinal tract (stomach, duodenum and jejunum), rectum, and sigmoid colon (enema) during B-ultrasound, CT, and magnetic resonance examinations. Parenchymal organs, retroperitoneal structures, delineation of lesions, elimination of gas and artifacts, easy to use and low price are effective auxiliary means of imaging examination, and have good clinical application value.

3. Contrast agent characterization

Experimental methods: The magnetic test capsules were made, and the morphology of the composite nanoparticles was observed by SEM. Take a sample with an appropriate concentration, take a certain amount of solution and drop it on the PMMA film supported by the magnetic test, dry it and place it under the SEM instrument for observation. The magnetic properties of the nanosamples were measured at room temperature using a vibrating sample magnetometer (VSM), with the capsules immobilized in a copper rod and placed in a vibrating cavity.

Experimental results: The structure and morphology of the prepared contrast agent composite nanoparticles were characterized by SEM. The nanoparticles in the capsules were spherical in appearance and had good uniformity and dispersion. Select the positive and negative magnetic field range +2 Tesla (T) to -2 Tesla (T) for sweeping test, the test step size is set to 50 Oersteds (Oe), and the measurement temperature is 300 Calvin (K). The test results are shown in the figure. It can be seen that the magnetization curve is S-shaped on the applied magnetic field, and the residual magnetization and coercive field are extremely low. When the external magnetic field is removed, the sample does not retain the residual magnetization; the saturation magnetization can be as high as 73.5 emu/g, indicating that the nanosamples exhibit typical superparamagnetic behavior at room temperature.

4. Description of contrast agent in vitro MRI.

Experimental method: The nanoparticles were prepared into an aqueous solution with a concentration of 0.05-1 ^{mmol·L -1} , and were neatly arranged on the test tube rack. T2-weighted magnetic resonance images were obtained using a magnetic resonance imager, and T2 values were recorded. Parameter setting: repetition time (TR)=2000ms, field of view (FOV)=200mm, echo time (TE)=40ms. Taking Fe ion concentration as abscissa and 1/T2 value as ordinate, draw a linear graph.

Experimental results: The possibility of the synthesized nanoparticles as T2-type magnetic resonance imaging contrast agents was investigated by in vitro magnetic resonance imaging. According to the experimental results, with the increase of the nanoparticle concentration, the magnetic resonance signal intensity decreases significantly, which is reflected in the image as the magnetic resonance imaging target area getting darker and darker. In addition, the 1/T2 value was linearly fitted with the Fe ion concentration, and it was found that 1/T2 had a good linear relationship with the Fe ion concentration, and the transverse relaxation rate was 25.44 mmol·L ^-1 s ^-1 , indicating that the nanoparticles have Good in vitro magnetic resonance imaging performance, can be used as in vitro T2-weighted MRI contrast agent.

5. Describe the establishment of the nude mouse Luc-U87-MG orthotopic glioblastoma model.

Experimental method: BAL B/c nude mice aged 5-6 weeks and weighing about 20 g were used to establish the model. First, Luc-U87-MG cells were cultured in high glucose DMEM containing 10% fetal bovine serum and 1% penicillin/streptomycin under the conditions of 37°C and 5% CO ₂ . When the cells grew well, trypsinization was performed. The cells were collected for in situ glioblastoma inoculation, and each mouse was inoculated with a concentration of 5×10 ⁴ cells (5 μL of single cell suspension). The anesthetic sodium pentobarbital (60 μg/kg) was injected according to the weight of the mice, and after the mice were completely anesthetized, the mice were fixed in a stereotaxic apparatus. After disinfection with iodine and alcohol, the head skin was cut longitudinally to expose the skull. The anterior fontanelle is the origin, and the drilling is positioned 2mm and 1mm forward. The Luc-U87-MG cell suspension was absorbed with a 5 μL microsyringe, the hole was drilled down 3 mm, the needle was withdrawn by 1 mm, and the cells were injected at a speed of 0.5 μL/min. After all injections, stop the needle for 10 minutes and withdraw the needle. After the wound was disinfected, the skin was sutured with medical sutures and disinfected, and the mice were continuously observed after operation. 3 weeks after surgery, use a small animal in vivo imager to observe and confirm the success of the model.

Experimental results: 3 weeks after in situ inoculation of Luc-U87-MG cells, the successful inoculation was detected by a small animal in vivo imager. In mice that were not successfully inoculated, the tumor cells were not concentrated at the intracranial injection site and migrated below the head and neck, while in successfully inoculated nude mice, tumor cells were observed to accumulate at the intracranial injection site. tumor.

6. Explain in vivo magnetic resonance T2-weighted imaging and SWI (T2*) imaging.

Experimental methods: The tumor-bearing nude mice with successfully established in situ glioblastoma were selected for magnetic resonance imaging studies. Magnetic resonance imaging was performed using Siemens Magnetom Trio 3.0T magnetic resonance apparatus and mouse coils. Imaging animals were divided into 2 groups of 5:

Experimental imaging group (group 1): a contrast agent was injected into the tail vein at a dose of 10 mg Fe ₃ O ₄ /kg.

Negative control group (group 2): Physiological saline was injected into the tail vein at the same dose. The naked mice were anesthetized with isoflurane for the first scan and images were collected. After the probe was injected, enhanced scanning was performed and images were collected. The time points were 0h, 2h, 4h, and 24h. Scan parameters:

T2-weighted imaging: TE=93ms; TR=3000ms; slice thickness (SL)=1mm; field of view (FoV)=66mm×66mm; matrix size=256×256, NEX: 5;

SWI (T2*) imaging: TE=20ms; TR=32ms; slice thickness (SL)=1mm; field of view (FoV)=50mm×50mm; matrix size=320×320, NEX:2.

Experimental results: Nude mice that successfully established in situ glioblastoma were selected for in vivo imaging. T2-enhanced scanning of tumor-bearing nude mice after injection of normal saline showed that normal saline could not effectively enhance and develop the tumor site on T2 imaging, but after injection of contrast agent, the tumor site was brighter and more visible on T2 imaging. The enhancement effect diminishes over time. Since the tumor itself presents bright signal in T2 plain scan, and normal brain tissue presents dark signal, the contrast agent has obvious advantages in T2 enhancement of intracranial tumors.

Susceptibility-weighted imaging (SWI, T2*) is a magnetic resonance imaging technique that uses different tissue susceptibility to image. It is very sensitive to local magnetic field changes and appears as low signal on the image. Because iron deposition can cause magnetic field changes, SWI technology was used in the detection of brain tumors using SPIONs probes modified with targeting peptides, and compared with T2-weighted imaging. The results show that SWI can clearly outline the tumor contour and enhance the contrast between normal tissue and tumor tissue.

At 0 min after SWI injection (in actual operation, a T2 sequence was scanned for 4.5 min, and a SWI sequence was scanned for 7.5 min), obvious signal changes in the tumor part could be seen, and the dark signal would gradually weaken over time. After 24 hours, SPIONs can still be detected at the tumor site by SWI sequence scanning, but T2-weighted imaging cannot be monitored for a long time. In the tumor-bearing nude mice injected with contrast agent, the retention of contrast agent could be detected in the glioblastoma site for a long time (24h).

7. Describe the fluorescence imaging of isolated organs.

Experimental methods: Bioluminescence imaging and near-infrared fluorescence imaging of tumor-bearing nude mice successfully established in situ glioblastoma were performed using in vivo imaging of small animals. Near-infrared fluorescence imaging of intracranial tumors requires craniotomy, therefore, ex vivo imaging is used. The experimental grouping was the same as "2. Contrast agent abdominal magnetic resonance MRI assessment.", and were divided into 2 groups. Twenty-four hours after the tail vein material injection, a certain amount of luciferin potassium salt solution (150 mg/kg) was intraperitoneally injected, and after 15 minutes of reaction, the experimental animals were perfused with normal saline and 4% paraformaldehyde, and their brain tissues and other tissues were collected. The main organs (heart, liver, spleen, lung, kidney) were then used to observe the in situ imaging of brain glioblastoma and the distribution of materials in each organ by a small animal in vivo imaging system (the excitation wavelength for near-infrared fluorescence imaging was set at 720 nm, the emission wavelength of 820 nm was detected).

Experimental results: The near-infrared fluorescence imaging effect of contrast agents was evaluated by in vitro fluorescence imaging with a small animal in vivo imager. 24h after the probes in the normal saline control group and the contrast agent experimental group were injected into the tumor-bearing nude mice, the size of the brain glioblastoma in different groups was detected by bioluminescence imaging (BLI), and the probes in different groups were detected by near-infrared fluorescence imaging. The amount of aggregation at the tumor site. Finally, the mean fluorescence intensity (total fluorescence intensity/bioluminescence photon number) was calculated to evaluate the mean aggregation of probes at the tumor site. The results showed that the average fluorescence intensity of the contrast agent experimental group at the tumor site was significantly higher than that of the control group, and the difference was significant (p<0.05).

8. Explain the localization of the contrast agent in the tumor-bearing brain

Experimental method: After scanning, the experimental animals were perfused with normal saline and 4% paraformaldehyde, and their brain tissue was taken, and fixed with fixative for 2 days, and then transferred to 30% sucrose solution for dehydration. 3 times, after complete dehydration, the brain tissue was embedded with OCT embedding medium and placed in a cryostat for frozen sectioning. During sectioning, the sagittal plane of the brain was kept parallel to the blade, and the thickness was 8 μm. Remove the slides from the slides, rinse the sections with water for 2 min after drying, and then rinse with ultrapure water for 2 min. Mix equal volumes of Prussian blue solution A (2% HCl solution) and solution B (2% potassium ferrocyanide solution) to prepare a dye solution, incubate the tissue sections for 30 minutes, and then wash with ultrapure water twice, 3 minutes each time . Add counterstain solution (2% nuclear fast red dye) for 1 min. After dyeing, wash with pure water, air dry, seal with neutral resin, and observe the specific distribution of probes in the brain by optical microscope.

Experimental results: After the above-mentioned fixed brain tissue was further sliced, Prussian blue was used to detect the distribution of probes in the brain and tumor locations. By staining analysis, the contrast agent was aggregated at the tumor margin, and the results were consistent with the MR imaging results, and the probe could effectively aggregate at the tumor margin.

9. Description of the tissue distribution of probes in tumor-bearing mice

Experimental method: Take healthy BALB/ _c mice and randomly divide them into ₂ groups, which are set as a normal saline control group and a contrast agent experimental group, with 5 mice in each group. An equal volume of normal saline was injected into the corresponding mice, and they were sacrificed after 24 hours. The organs (brain, heart, liver, spleen, lung, and kidney) of 2 mice in each group were randomly taken and fixed, and 0.2 g of each organ was cut into Crushed, add 1 mL of hydrogen peroxide, 2 mL of nitric acid to digest for two days, oil bath at 120 °C until there is no precipitation, after evaporation to dryness, add 5 mL of 2% HNO ₃ solution to the volume, after sonication for 20 min, take 4 mL of the solution into an EP tube, use Fe content was determined by ICP-MS method.

Experimental results: The distribution and content of probes in major organs in vivo were determined by ICP-MS and fluorescence analysis. The main organs (heart, liver, spleen, lung, kidney, brain) of the mice were collected by perfusion 24 hours after the probe injection for in vitro fluorescence imaging with a small animal in vivo imager. The results of total fluorescence intensity analysis showed that the contrast agent should be concentrated in the two major metabolic organs of liver and kidney. Through further quantitative analysis by ICP-MS, although the total amount of nanoprobes has obvious aggregation in the liver and kidney, but from the Fe mass fraction analysis, the Fe mass ratio of the spleen is the highest, which may be related to its role as an iron metabolism organ. Certain relationship.

10. Description of the cytotoxicity of contrast agents

Experimental methods: The cytotoxicity of contrast agents was investigated by MTT assay. MCF-7 cells in logarithmic growth phase were taken and seeded in 96-well plates at 5×10 ³ cells per well, and then placed in a cell culture incubator at 37°C with 5% CO ₂ . After 24 h, the original medium was replaced with 100 μL of fresh medium containing contrast agents with different mass concentrations (10, 20, 50, 100, 200, 500 and 1 000 μg·mL ^-1 ), and the culture was continued for 24 h. The culture medium was aspirated again and washed three times with PBS, and then 100 μL of MTT (1 mg·mL ^-1 ) was added to each well and incubated for 4 h before removing. Add 150 μL of dimethyl sulfoxide to each well, shake for 10 min, and use a microplate reader to measure the absorbance at 570 nm of each well. Six replicate wells were set for each concentration, and the experiment was repeated three times.

Experimental results: MTT method was used to study the toxic effect of contrast agent on MCF-7 cells. After 24h incubation, the survival rate of MCF-7 cells decreased with the increase of contrast agent concentration. However, the survival rate of MCF-7 cells was not significantly reduced at each concentration. When the concentration of contrast agent was less than 200 μg·mL ^-1 , the survival rate was above 90%, and when the concentration was increased to 5 times, it still showed The cell survival rate was nearly 85%, indicating that the contrast agent has good biocompatibility and can be used for subsequent in vivo studies.

11. Describe the preliminary in vivo safety assessment of contrast agents.

Experimental method: Healthy BAL B/c mice were randomly divided into 4 groups, divided into a normal saline control group and an experimental group, with 9 mice in each group. The probe solution or an equal volume of normal saline was injected into the mice through the tail vein at a dose of 10 mg of contrast agent. Three mice in each group were sacrificed on the 1st, 3rd, and 7th days after the injection, and their organs were harvested. (heart, liver, spleen, lung, kidney) were fixed, followed by paraffin sectioning, hematoxylin-eosin (HE) staining, and light microscopy for histopathological observation.

Experimental results: The contrast agent was injected into the tail vein. After the 1st, 3rd, and 7th days, the HE staining results of the main organs (heart, liver, spleen, lung, and kidney) in different groups were analyzed. Compared with the control group, the experimental group had no obvious pathological features. This indicates that the constructed probes do not have long-term systemic toxicity in vivo and have good in vivo safety.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims (10)

1. a magneto-optical dual-field contrast agent, is characterized in that, the structure of described contrast agent is the superparamagnetic iron tetroxide structure that hyaluronic acid wraps mesoporous silica coating; Simultaneously, mesoporous silica The coated superparamagnetic iron tetroxide structure is also filled with rare earth elements. 2 . The magneto-optical dual-field contrast agent according to claim 1 , wherein the rare earth material is erbium. 3 . 3. a preparation method of magneto-optical dual-field contrast agent, is characterized in that, comprises: (1), prepare superparamagnetic iron tetroxide: add ammonia water quickly to the mixed solution of FeCl ₃ and FeCl ₂ , continue the reaction and finish and be cooled to room temperature, and obtain superparamagnetic iron tetroxide after cleaning; (2), the preparation of mesoporous silica coating layer: stirring and mixing cetyl trimethyl ammonium bromide, absolute ethanol, deionized water, adding ammonia solution and superparamagnetic ferric tetroxide simultaneously, After stirring, wait for a period of time and continue to add ethyl orthosilicate dropwise, and the mixture is heated in a water bath for the first time to obtain a precipitate; the precipitate is centrifuged to obtain a secondary precipitate, the secondary precipitate is washed, and the supernatant is poured out. Then NH ₄ NO ₃ and ethanol solution were added and stirred, and the mixed solution was heated in a water bath for the second time to obtain three precipitates. After standing for a period of time, the supernatant liquid was poured out and washed; the final turbid liquid was Drying is carried out to finally obtain a superparamagnetic iron tetroxide structure coated with mesoporous silica; (3) Mesoporous filling with rare earth elements: put the prepared mesoporous silica-coated superparamagnetic ferric oxide structure and erbium nitrate solution in a vortex mixer and then dry them thoroughly. Finally, a superparamagnetic ferric oxide doped rare earth element structure coated with mesoporous silica is obtained; (4), mixing the product prepared in (3) with hyaluronic acid and water to obtain a magneto-optical dual-field contrast agent. 4. the preparation method of a kind of magneto-optical dual-field contrast agent as claimed in claim 3, is characterized in that, in step (1), FeCl ₃ , FeCl ₂ and ammonia water volume are respectively 10-30ml, 1-30ml, 4 -10ml; preferably, 20ml, 20ml, 7.5ml; or, the FeCl concentration is 0.1-0.3mol/ _L ; preferably, it is _0.2mol /L; or, the FeCl concentration is 0.05-0.2mol /L; preferably 0.1 mol/L; or, the weight percentage of the ammonia water is 28wt%. 5. The preparation method of a magneto-optical dual-field contrast agent according to claim 3, wherein in step (1), after FeCl ₃ and FeCl ₂ are mixed, stir for 5-15min, preferably 10min; Or, the continuous reaction time is 15-35min, preferably 25min; Or, what is used for cleaning is sodium chloride and double distilled water, and the volume ratio of sodium chloride solution and double distilled water is 1:2～3; Or, chlorine The concentration of the sodium chloride solution is 0.01-0.03 mol/L, preferably 0.02 mol/L; or, drying: the heating and drying temperature is 60° C., and the time is 1-2 hours. 6. the preparation method of a kind of magneto-optical dual-field contrast agent as claimed in claim 3, is characterized in that, in step (2), the quality of cetyl trimethyl ammonium bromide is 60-85mg, anhydrous The volume ratios of ethanol and deionized water are respectively 10-20ml and 20-35ml; preferably, the mass of cetyltrimethylammonium bromide is 75mg, and the volume ratios of absolute ethanol and deionized water are respectively 15ml, 25ml; or, the dosages of ammonia solution, ferric tetroxide nanoparticles and ethyl orthosilicate are respectively 200-300μl, 0.15-0.40g, 100-150μl; preferably, 250μL: 0.23g: 129μL. 7. The preparation method of a magneto-optical dual-field contrast agent according to claim 3, wherein in step (2), the concentration of the ammonia solution is 28wt%; or, the waiting time for the end of stirring is 5-15s , preferably 10s; or, the first water bath heating temperature is 40-70°C, preferably 60°C; or, the first water bath heating time is 4-7h, preferably 6h; or, NH ₄ NO ₃ ethanol The solution volume is 10-30ml, preferably 20ml; or, the second water bath heating and stirring time is 1-3h, and the temperature is 50-70 ° C; or, cleaning: absolute ethanol and deionized water volume ratio is 1: 2 to 3; drying: the time is 2h, and the temperature is 80°C. 8. Application of the magneto-optical dual-field contrast agent according to claim 1 or 2 and/or the contrast agent obtained by the preparation method of the magneto-optical dual-field contrast agent according to any one of claims 3-7 in magnetic resonance imaging. 9. The contrast agent obtained by the magneto-optical dual-field contrast agent of claim 1 or 2 and/or the preparation method of the magneto-optical dual-field contrast agent of any one of claims 3-7 is used in coronary intravascular high-definition angiography Applications. 10. In the hemodynamic evaluation of the contrast agent obtained by the magneto-optical dual-field contrast agent according to claim 1 or 2 and/or the preparation method of the magneto-optical dual-field contrast agent according to any one of claims 3-7 application.

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